LabDisk Zentrifugale Mikrofluidik zur vollintegrierten

LabDisk
Zentrifugale Mikrofluidik zur vollintegrierten
Automatisierung von Laborabläufen
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Outline
 Motivation
 Introduction to centrifugal microfluidics
 Application example 1:
Detection of Bacillus anthracis & Francisella tularensis
 Application example 2:
Detection of Botulinum neurotoxin
 Conclusion
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Time critical situations…
Bio-terrorism
Environmental disasters
require
rapid decision making
Severe infections
Fertility testing
Bioprocess control
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
Plant protection
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Solution
Analysis at the point-of-care
 Results within minutes instead of days
 Automation of laboratory processes by untrained personel
20 mm
50 mm
Portable device
~ 2 kg; ~ DIN A4 Footprint
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
Low-cost polymer disposable
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Centrifugal microfluidics
Forces for liquid actuation and manipulation
Advantages of centrifugal microfluidics:
acceleration
 (Pulse free) liquid actutation by rotation
Euler force
centrifugal force
 No connection to external pressure sources
 No integration of active components
LabDisk at rest
Coriolis force
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
capillary force
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Modular fluidic implementation
using established and validated unit-operations
Valving
Metering
Aliquoting
Mixing
Reaction &
Detection
Reagent storage
Venting
Filling
Center of
rotation
Modular
fluidic design
F >F
using unit operations
Z2
Z1
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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DNA based pathogen detection
Example: Bacillus anthracis & Francisella tularensis
 Motivation:
 B. anthracis and F. tularensis
misused as bioweapon
 Causative agents of letal infections
B. anthracis
 Anthrax
F. tularensis
 Tularämie
 Fast detection at the point-of-care
required to initiate counteraction
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Some recent incidents…..
 Several letters containing B. Anthracis spores send to several
institutions in the US following after 9/11
 22 people were infected with Anthrax  5 people died
Source: http://de.wikipedia.org/wiki/Bioterrorismus
 Not only in the US…
Source: Badische Zeitung; 14. August 2014
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Implementation of pathogen detection
 Microfluidic Solution for automated pathogen detection:
 LabDisk with
Center
of rotation
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Implementation of pathogen detection
 Microfluidic Solution for automated pathogen detection:
 LabDisk with integrated cell lysis & DNA extraction…
Wash 2
Wash 1
Lyse
& bind
O. Strohmeier, LabChip, 2013, 13, 146
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Implementation of pathogen detection
 Microfluidic Solution for automated pathogen detection:
 LabDisk with integrated cell lysis & DNA extraction…
 … mix extracted DNA with RPA polymerase
Mix
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Implementation of pathogen detection
 Microfluidic Solution for automated pathogen detection:
 LabDisk with integrated cell lysis & DNA extraction…
 … mix extracted DNA with RPA polymerase
 … aliquoting and real-time amplification (isothermal RPA)
D. Mark, Microfluid Nanofluid, 2010, 10, 1279
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Results
DNA based Sample-to-answer detection
 Successful detection of 6 x 104 and 6 x 106 genome equivalents of
B. anthracis and F. tularensis, from 200 µL blood plasma samples
 Time to result < 45 min (35 min lysis & extraction, 10 min amp.)
~ 10 min
F. tularensis
B. anthracis
Negative control
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Outlook
 Diversification of pathogen pannels for detection of:
 Respiratory infections:
 Tropical fevers:
(Para-) Influenza / Coronavirus etc.  23 targets
Malaria / Dengue / Chikunguya etc.
 Integration of on-board
liquid reagent prestorage
T. van Oordt, Lab Chip, 2013, 13, 2888
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Protein detection
Example: Botulinum neurotoxin
 Motivation
 Botulinum neurotoxin (BoNT)
is strong toxin
 Causes botulismus disease
 Intoxication from food, mainly
spoiled meat and vegetables
 BoNT is potential bioweapon
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Assay principle
 Luciferase Reporter Assay
 Luciferase linked to beads with peptide linker
 BoNT cleaves peptide linker…
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Assay principle
 Luciferase Reporter Assay
 Luciferase is released and beads are removed
 Released Luciferase is merged with luciferin
T. van Oordt, RSC Advances, 2013, 3, 22046
G.B. Stevens, Analyst, 2013, 138, 6154
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Implentation of BoNT detection
 One LabDisk with 7 structures for 7 parallel Tests
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Implentation of BoNT detection
 One LabDisk with 7 structures for 7 parallel Tests
 Sample & reagents added to Disk
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Implentation of BoNT detection
 One LabDisk with 7 structures for 7 parallel Tests
 Sample & reagents added to Disk
 After incubation, priming buffer actuates liquid into detection chamber…
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Implentation of BoNT detection
 One LabDisk with 7 structures for 7 parallel Tests
 Sample & reagents added to Disk
 After incubation, priming buffer actuates liquid into detection chamber…
 Where the sample with the released Luciferase is mixed with substrate
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Implentation of BoNT detection
 One LabDisk with 7 structures for 7 parallel Tests
 Sample & reagents added to Disk
 After incubation, priming buffer actuates liquid into detection chamber…
 Where the sample with the released Luciferase is mixed with substrate
 Detection of luminescence signal
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Results
Detection of Botulinum neurotoxin
 Tested toxin: Botulinum neurotoxin Type A
 As light chain (LC) in buffer (9 pM – 2 nM)
 As full toxin in buffer and whole milk (8 pM – 6 nM)
Intra-Day: 1 – 13 %
Inter-Day: 1 – 7 %
Inter-Assay: 10 - 13 %
T. van Oordt, RSC Advances, 2013, 3, 22046
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Conclusion
 Centrifugal microfluidic LabDisk suitable
for automation of laboratory assays (at the point-of-care)
 Processing in portable device at the point-of-care
 Low-cost fabrication of cartridge from thin polymer foils
 Application example 1:
 Nucleic acid based detection of B. anthracis (6 x 104 geq.)
and F. tularensis (6 x 106 geq.) from blood plasma
 Time-to-result < 45 minutes
 Application example 2:
 Detection of Botulinum neurotoxin as light chain (9 pM – 2 nM) and
full toxin from buffer and whole milk (8 pM – 6nM)
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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Outlook
 Expansion of prototpying capacity
 Capacity: > 50.000 LabDisks / year
 Investigation of production up-scaling
 Production of LabDisks
e.g. for clinical validation studies
Forming
Filling
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
Sealing
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Thank you for you attention !
Parts of this work were funded
by the BMBF within the Project
S.O.N.D.E (13N10116)
For any interest in collaboration:
[email protected]
Oliver Strohmeier - 23.09.2014 - Heiligenstädter Kolloqium 2014
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